US 7880468 B2 Abstract The present invention presents a new approach to rapidly obtaining precise high-dimensional NMR spectral information, named “GFT NMR spectroscopy”, which is based on the phase sensitive joint sampling of the indirect dimensions spanning a subspace of a conventional NMR experiment. The phase-sensitive joint sampling of several indirect dimensions of a high-dimensional NMR experiment leads to largely reduced minimum measurement times when compared to FT NMR. This allows one to avoid the “sampling limited” data collection regime. Concomitantly, the analysis of the resulting chemical shift multiplets, which are edited by the G-matrix transformation, yields increased precision for the measurement of the chemical shifts. Additionally, methods of conducting specific GFT NMR experiments as well as methods of conducting a combination of GFT NMR experiments for rapidly obtaining precise chemical shift assignment and determining the structure of proteins or other molecules are disclosed.
Claims(8) 1. A method for assigning chemical shift values of γ-, δ-, and ε-aliphatic sidechain protons,
^{1}H^{γ/δ/ε}, and chemical shift values of γ-, δ-, and ε-aliphatic sidechain carbons located peripheral to β-carbons, ^{13}C^{γ/δ/ε}, of a protein molecule comprising:
providing a protein sample;
conducting a set of Fourier transformation (FT) nuclear magnetic resonance (NMR) experiments on the protein sample comprising: (1) a (5,3)D[
HCC,CH—COSY] FT NMR experiment to measure and connect the chemical shift values of a proton of amino acid residue i−1, ^{1}H_{i−1}, a carbon of amino acid residue i−1 coupled to ^{1}H_{i−1}, ^{13}C_{i−1}, a carbon coupled to ^{13}C_{i−1}, ^{13}C_{i−1} ^{coupled}, and a proton coupled to ^{13}C_{i−1} ^{coupled}, ^{1}H_{i−1} ^{coupled}, and (2) a (5,3)D [HBHACBCACA(CO)NHN] FT NMR experiment to measure and connect the chemical shift values of α- and β-protons of amino acid residue i−1, ^{1}H^{α/β} _{i−1}, and α- and β-carbons of amino acid residue i−1, ^{13}C^{α/β} _{i−1}; andobtaining assignments of the chemical shift values of
^{1}H^{γ/δ/ε} and ^{13}C^{γ/δ/ε} by (i) identifying ^{1}H_{i−1}, ^{13}C_{i−1}, ^{13}C_{i−1} ^{coupled}, and ^{1}H_{i−1} ^{coupled }measured by said (5,3)D [HCC,CH—COSY] FT NMR experiment as ^{1}H^{α} _{i−1}, ^{13}C^{α} _{i−1}, ^{13}C^{β} _{i−1}, and ^{1}H^{β} _{i−1}, respectively, and thereby matching the chemical shift values of ^{1}H^{α/β} _{i−1 }and ^{13}C^{α/β} _{i−1 }with the chemical shift values of ^{1}H^{α/β} _{i−1 }and ^{13}C^{α/β} _{i−1 }measured by said (5,3)D [HBHACBCACA(CO)NHN] FT NMR experiment, and (ii) using the chemical shift values of ^{1}H^{α/β} _{i−1 }and ^{13}C^{α/β} _{i−1 }in conjunction with other chemical shift connections from said (5,3)D [HCC,CH—COSY] FT NMR experiment to measure the chemical shift values of ^{1}H^{γ/δ/ε} _{i−1 }and ^{13}C^{γ/δ/ε} _{i−1}.2. The method according to
subjecting the protein sample to nuclear Overhauser enhancement spectroscopy (NOESY) to deduce the tertiary structure of the protein molecule.
3. The method according to
subjecting the protein sample to NMR experiments that measure scalar coupling constants to deduce the tertiary structure of the protein molecule.
4. The method according to
subjecting the protein sample to NMR experiments that measure residual dipolar coupling constants to deduce the tertiary structure of the protein molecule.
5. A method for assigning chemical shift values of γ-, δ-, and ε-aliphatic sidechain protons,
^{1}H^{γ/δ/ε}, and chemical shift values of γ-, δ-, and ε-aliphatic sidechain carbons located peripheral to β-carbons,^{13}C^{γ/δ/ε}, of a protein molecule comprising:
providing a protein sample;
conducting a set of Fourier transformation (FT) nuclear magnetic resonance (NMR) experiments on the protein sample comprising: (1) a (4,2)D [
HCCH—COSY] FT NMR experiment to measure and connect the chemical shift values of a proton of amino acid residue i−1, ^{1}H_{i−1}, a carbon of amino acid residue i−1 coupled to ^{1}H_{i−1}, ^{13}C_{i−1}, a carbon coupled to ^{13}C_{i−1}, ^{13}C_{i−1} ^{coupled}, and a proton coupled to ^{13}C_{i−1} ^{coupled}, ^{1}H_{i−1} ^{coupled},and (2) a (5,3)D [HBHACBCACA(CO)NHN] FT NMR experiment to measure and connect the chemical shift values of α- and β-protons of amino acid residue i−1, ^{1}H^{α/β} _{i−1}, and α- and β-carbons of amino acid residue i−1, ^{13}C^{α/β} _{i−1}; andobtaining assignments of the chemical shift values of
^{1}H^{γ/δ/ε} and ^{13}C^{γ/δ/ε} by (i) identifying ^{1}H_{i−1}, ^{13}C_{i−1}, ^{13}C_{i−1} ^{coupled}, and ^{1}H _{i−1} ^{coupled }measured by said (4,2)D [HCCH—COSY] FT NMR experiment as ^{1}H^{α} _{i−1}, ^{13}C^{α} _{i−1}, ^{13}C^{β} _{i−1}, and ^{1}H^{β} _{i−1}, respectively, and thereby matching the chemical shift values of ^{1}H^{α/β} _{i−1} and ^{13}C^{α/β} _{i−1} with the chemical shift values of ^{1}H^{α/β} _{i−1} and ^{13}C^{α/β} _{i−1} measured by said (5,3)D HBHACBCACA(CO)NHN] FT NMR experiment, and (ii) using the chemical shift values of ^{1}H^{α/β} _{i−1} and ^{13}C^{α/β} _{i−1} in conjunction with other chemical shift connections from said (4,2)D [HCCH—COSY] FT NMR experiment to measure the chemical shift values of ^{1}H^{γ/δ/ε} _{i−1} and ^{13}C^{γ/δ/ε} _{i−1}.6. The method according to
subjecting the protein sample to nuclear Overhauser enhancement spectroscopy (NOESY) to deduce the tertiary structure of the protein molecule.
7. The method according to
subjecting the protein sample to NMR experiments that measure scalar coupling constants to deduce the tertiary structure of the protein molecule.
8. The method according to
subjecting the protein sample to NMR experiments that measure residual dipolar coupling constants to deduce the tertiary structure of the protein molecule.
Description This application is a division of U.S. patent application Ser. No. 10/973,807 filed Oct. 26, 2004, which is a division of U.S. patent application Ser. No. 10/617,482, filed Jul. 11, 2003, now U.S. Pat. No. 6,831,459, which claims the benefit of U.S. Provisional Patent Application Ser. Nos. 60/395,591, filed Jul. 11, 2002, and 60/441,385, filed Jan. 16, 2003, which are hereby incorporated by reference in their entirety. This invention arose out of research sponsored by the National Science Foundation (Grant No. MCB 0075773) and National Institutes of Health (Grant No. P50 GM62413-01). The U.S. Government may have certain rights in this invention. The present invention relates to methods of using G-matrix Fourier transformation nuclear magnetic resonance (GFT NMR) spectroscopy for rapidly obtaining and connecting precise chemical shift values and determining the structure of proteins and other molecules. Nuclear magnetic resonance (NMR) (Ernst et al., An increase in dimensionality is, however, limited by the need to independently sample the indirect dimensions, because this leads to longer measurement times. Although the measurement time can be somewhat reduced by aliasing signals (Cavanagh et al., In view of these considerations, “sampling” and “sensitivity limited” data collection regimes are defined (Szyperski et al., In general, phase-sensitive acquisition of an N-dimensional (ND) FT NMR experiment (Ernst et al., Thus, higher-dimensional FT NMR spectroscopy suffers from two major drawbacks: (i) The minimal measurement time of an ND FT NMR experiment, which is constrained by the need to sample N−1 indirect dimensions, may exceed by far the measurement time required to achieve sufficient signal-to-noise ratios. (ii) The low resolution in the indirect dimensions severely limits the precision of the indirect chemical shift measurements. The present invention is directed to overcoming the deficiencies in the art. The present invention relates to a method of conducting a (N,N−K) dimensional (D) G-matrix Fourier transformation (GFT) nuclear magnetic resonance (NMR) experiment where N is the dimensionality of an N-dimensional (ND) Fourier transformation (FT) NMR experiment and K is the desired reduction in dimensionality relative to N. The method involves providing a sample and applying radiofrequency pulses for the ND FT NMR experiment to the sample. Then, m indirect chemical shift evolution periods of the ND FT NMR experiment are selected, where m equals K+1, and the m indirect chemical shift evolution periods are jointly sampled. Next NMR signals detected in a direct dimension are independently cosine and sine modulated to generate (N−K)D basic NMR spectra containing frequency domain signals with 2 Another aspect of the present invention relates to a method for sequentially assigning chemical shift values of an α-proton, Yet another aspect of the present invention relates to a method for sequentially assigning chemical shift values of an α-proton, A further aspect of the present invention relates to a method for sequentially assigning chemical shift values of α- and β-carbons, The present invention also relates to a method for sequentially assigning chemical shift values of α- and β-carbons, Another aspect of the present invention relates to a method for assigning chemical shift values of γ-, δ-, and ε-aliphatic sidechain protons, Yet another aspect of the present invention relates to a method for assigning chemical shift values of γ-, δ-, and ε-aliphatic sidechain protons, A further aspect of the present invention relates to a method for assigning chemical shift values of a γ-carbon, The present invention also relates to a method for assigning chemical shift values of aliphatic and aromatic protons and aliphatic and aromatic carbons of an amino acid residue containing aliphatic and aromatic spin systems in a protein molecule. The method involves providing a protein sample and conducting a set of G matrix Fourier transformation (GFT) nuclear magnetic resonance (NMR) experiments on the protein sample including: (1) a first GFT NMR experiment which is selected from the group consisting of a (5,3)D [ Another aspect of the present invention relates to a method for obtaining assignments of chemical shift values of The present invention discloses a number of specific GFT NMR experiments and different combinations of those experiments which allows one to obtain sequential backbone chemical shift assignments for determining the secondary structure of a protein molecule and complete assignments of chemical shift values for a protein molecule including aliphatic and aromatic sidechain spin systems. The present invention provides a generally applicable approach for NMR data acquisition and processing named “GFT NMR spectroscopy”. This approach is based on the phase-sensitive joint sampling of several indirect dimensions while ensuring that all chemical shift correlations are retained. The employment of GFT NMR focuses on the sampling limited data collection regime and, considering that NMR measurements longer than about a week are impracticable, on the acquisition of five- or higher-dimensional spectral information. GFT NMR relaxes on constraints arising from two major drawbacks of FT NMR, that is, the problem of having excessive or prohibitively long measurement times due to sampling of indirect dimensions and the limited precision of chemical shift measurements in the indirect dimensions arising from comparably low digital resolution. Within a few hours or less, GFT NMR spectroscopy affords the correlations of even five- or higher-dimensional FT NMR spectra acquired with high digital resolution. Thus, GFT NMR spectroscopy allows one to tune measurement times to sensitivity requirements without compromising on the dimensionality or the digital resolution. High-throughput efforts such as NMR-based structural genomics (Montelione et al., In the sensitivity limited regime, GFT NMR may be advantageous in cases where an extended radiofrequency (rf) phase cycle is desirable for spectral editing and/or improved artifact suppression (Cavanagh et al., _{1}, Ω_{2 }or Ω_{3 }is added to or subtracted from Ω_{0 }in all of the four selected spectra (i.e., no splitting is present among the four selected spectra which encodes the respective chemical shift). The spectra selected for a particular combination number are indicated as dots. The statistical model used for the Monte Carlo simulations is the same as described in the legend of - 1: Ω
_{0}(^{13}C_{i−1}^{α})+Ω_{1}(^{13}C_{i−1}^{β}); - 2: Ω
_{0}(^{13}C_{i}^{α})+Ω_{1}(^{13}C_{i}^{β}) - 3: Ω
_{0}(^{13}C_{i}^{α})+Ω_{1 }(^{13}C_{i}^{α}), Ω_{0}(^{13}C_{i−1}^{α})+Ω_{1}(^{13}C_{i−1}^{α}) - 4: Ω
_{0}(^{13}C_{i}^{α})−Ω_{1}(^{13}C_{i}^{α}) - 5: Ω
_{0}(^{13}C_{i−1}^{α})−Ω_{1}(^{13}C_{i−1}^{α}) - 6: Ω
_{0}(^{13}C_{i}^{α})−Ω_{1}(^{13}C_{i}^{β}) - 7: Ω
_{0}(^{13}C_{i−1}^{α})−Ω_{1}(^{13}C_{i−1}^{β}) - 8: Ω
_{0}(^{13}C_{i}^{α}) - 9: Ω
_{0}(^{13}C_{i−1}^{α})
- 1: Ω
_{0}(^{13}C^{α})+Ω_{1}(^{13}C^{α})+Ω_{2}(^{1}H^{α}) - 2: Ω
_{0}(^{13}C^{α})+Ω_{1}(^{13}C^{β})+Ω_{2}(^{1}H^{β}) - 3: Ω
_{0}(^{13}C^{α})+Ω_{1}(^{13}C^{α})−Ω_{2 }(^{1}H^{α}) - 4: Ω
_{0}(^{13}C^{α})+Ω_{1}(^{13}C^{β})−Ω_{2}(^{1}H^{β}) - 5: Ω
_{0}(^{13}C^{α})−Ω_{1}(^{13}C^{β})−Ω_{2}(^{1}H^{β}) - 6: Ω
_{0}(^{13}C^{α})−Ω_{1}(^{13}C^{α})−Ω_{2}(^{1}H^{α}) - 7: Ω
_{0}(^{13}C^{α})−Ω_{1}(^{13}C^{β})+Ω_{2}(^{1}H^{β}) - 8: Ω
_{0}(^{13}C^{α})−Ω_{1}(^{13}C^{α})+Ω_{2}(^{1}H^{α}) - 9: Ω
_{0}(^{13}C^{α})−Ω_{1}(^{13}C^{β}) - 10: Ω
_{0}(^{13}C^{α})−Ω_{1}(^{13}C^{α}) - 11: Ω
_{0}(^{13}C^{α})+Ω_{1}(^{13}C^{α}) - 12: Ω
_{0}(^{13}C^{α})+Ω_{1}(^{13}C^{β})
- 1: Ω
_{0}(^{13}C^{δ2})−Ω_{1}(^{13}C^{β})−Ω_{2}(^{1}H^{β}) - 2: Ω
_{0}(^{13}C^{δ2})−Ω_{1}(^{13}C^{β})+Ω_{2}(^{1}H^{β}) - 3: Ω
_{0}(^{13}C^{δ2})+Ω_{1}(^{13}C^{β})+Ω_{2}(^{1}H^{β}) - 4: Ω
_{0}(^{13}C^{δ2})+Ω(^{13}C^{β})−Ω_{2}(^{1}H^{β}) - 5: Ω
_{0}(^{13}C^{δ2})−Ω_{1}(^{13}C^{β}) - 6: Ω
_{0}(^{13}C^{δ2})+Ω_{1}(^{13}C^{β}) - 7: Ω
_{0 }(^{13}C^{δ2})
The present invention provides an NMR data acquisition scheme which is based on the phase sensitive joint sampling of the indirect dimensions spanning a subspace of a conventional NMR experiment. This allows one to very rapidly obtain high dimensional NMR spectral information. Since the phase-sensitive joint sampling yields subspectra containing “chemical shift multiplets”, alternative data processing is required for editing the components of the multiplets. The subspectra are linearly combined using a so-called “G-matrix” and subsequently Fourier transformed. The chemical shifts are multiply encoded in the resonance lines constituting the shift multiplets. This corresponds to performing statistically independent multiple measurements, and the chemical shifts can thus be obtained with high precision. To indicate that a combined G-matrix and FT is employed, the new approach is named “GFT NMR spectroscopy”. In GFT NMR spectroscopy, the chemical shift evolution periods spanning a given multidimensional subspace of an FT NMR experiment are “jointly” sampled ( A shift of φ The 2 The joint sampling of several indirect dimensions reduces the minimal measurement time, T GFT NMR spectroscopy combines (i) multiple phase sensitive RD NMR, (ii) multiple ‘bottom-up’ central peak detection, and (iii) (time domain) editing of the components of the chemical shift multiplets. The resulting formalism embodies a flexible, generally applicable NMR data acquisition scheme. Provided that m=K+1 chemical shift evolution periods of an ND experiments are jointly sampled in a single indirect “GFT dimension”, p=2 Thus, the present invention relates to a method of conducting a (N,N−K) dimensional (D) G-matrix Fourier transformation (GFT) nuclear magnetic resonance (NMR) experiment where N is the dimensionality of an N-dimensional (ND) Fourier transformation (FT) NMR experiment and K is the desired reduction in dimensionality relative to N. The method involves providing a sample and applying radiofrequency pulses for the ND FT NMR experiment to the sample. Then, m indirect chemical shift evolution periods of the ND FT NMR experiment are selected, where m equals K+1, and the m indirect chemical shift evolution periods are jointly sampled. Next NMR signals detected in a direct dimension are independently cosine and sine modulated to generate (N−K)D basic NMR spectra containing frequency domain signals with 2 As described earlier, the (N−K) D basic NMR spectra can be transformed into (N−K) D phase-sensitively edited basic NMR spectra by applying a G-matrix defined as In an alternate embodiment the method of conducting a (N,N−K)D GFT NMR experiment can further involve selecting m′ indirect chemical shift evolution periods of the (N−K)D FT NMR experiment where m′ equals K′+1. Then, the m′ indirect chemical shift evolution periods are jointly sampled. Next NMR signals detected in a direct dimension are independently cosine and sine modulated to generate (N−K−K′)D basic NMR spectra containing frequency domain signals with 2 In an alternate embodiment the method of conducting a (N,N−K)D GFT NMR experiment can further involve repeating one or more times the steps of selecting, jointly sampling, independently cosine and sine modulating, and transforming, where, for each repetition, the selecting involves selecting m-j indirect chemical shift evolution periods out of the m indirect chemical shift evolution periods, wherein j ranges from 1 to K, under conditions effective to generate 2 The method of conducting a (N,N−K)D GFT NMR experiment can also involve applying radiofrequency pulses of N-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) (Ernst et al., The present invention also discloses specific GFT NMR experiments and different combinations of those experiments which allows one to obtain sequential backbone chemical shift assignments for determining the secondary structure of a protein molecule and complete assignments of chemical shift values for a protein molecule including aliphatic and aromatic sidechain spin systems. Specific GFT NMR Experiments The present invention discloses the following six (N,N−K)D GFT NMR experiments for the assignment of polypeptide backbone and Thus, the present invention relates to the above method of conducting a (N,N−K)D GFT NMR experiment where N equals 5 and K equals 3 to conduct a (5,2)D [ The present invention also relates to the above method of conducting a (N,N−K)D GFT NMR experiment where N equals 5 and K equals 3 to conduct a (5,2)D [ Another aspect of the present invention relates to the above method of conducting a (N,N−K)D GFT NMR experiment where N equals 5 and K equals 2 to conduct a (5,3)D [ Yet another aspect of the present invention relates to the above method of conducting a (N,N−K)D GFT NMR experiment where N equals 5 and K equals 2 to conduct a (5,3)D [ A further aspect of the present invention relates to the above method of conducting a (N,N−K)D GFT NMR experiment where N equals 4 and K equals 1 to conduct a (4,3)D [ The present invention also relates to the above method of conducting a (N,N−K)D GFT NMR experiment where N equals 4 and K equals 1 to conduct a (4,3)D [ In addition, the present invention discloses the following GFT NMR experiments for the assignment of polypeptide backbone and sidechain resonances: (i) (4,3)D [HNN Thus, the present invention also relates to the above method of conducting a (N,N−K)D GFT NMR experiment where N equals 4 and K equals 1 to conduct a (4,3)D [HNN In an alternate embodiment the above method can be modified, where N equals 4 and K equals 2, to conduct a (4,2)D [HN In another alternate embodiment the above method can be modified, where N equals 4 and K equals 1 to conduct a (4,3)D [HNN(CO) In yet another alternate embodiment the above method can be modified, where N equals 4 and K equals 2 to conduct a (4,2)D [HN In another alternate embodiment the above method can be modified, where N equals 5 and K equals 2 to conduct a (5,3)D [HNN In yet another alternate embodiment the above method can be modified, where N equals 5 and K equals 3 to conduct a (5,2)D [HN Another aspect of the present invention relates to the above method of conducting a (N,N−K)D GFT NMR experiment where N equals 4 and K equals 1 to conduct a (4,3)D [ In an alternate embodiment the above method can be modified, where N equals 4 and K equals 2 to conduct a (4,2)D [ In another alternate embodiment the above method can be modified, where N equals 5 and K equals 2 to conduct a (5,3)D [ In yet another alternate embodiment the above method can be modified, where N equals 5 and K equals 3 to conduct a (5,2)D [ Another aspect of the present invention relates to the above method of conducting a (N,N−K)D GFT NMR experiment where N equals 5 and K equals 2 to conduct a (5,3)D [ In an alternate embodiment the above method can be modified, where N equals 6 and K equals 3 to conduct a (6,3)D [ In another alternate embodiment the above method can be modified, where N equals 5 and K equals 3 to conduct a (5,2)D [ In yet another alternate embodiment the above method can be modified, where N equals 6 and K equals 4 to conduct a (6,2)D [ Yet another aspect of the present invention relates to the above method of conducting a (N,N−K)D GFT NMR experiment where N equals 5 and K equals 2 to conduct a (5,3)D [ The present invention also relates to the above method of conducting a (N,N−K)D GFT NMR experiment where N equals 5 and K equals 2 to conduct a (5,3)D [ In an alternate embodiment the above method can be modified, where N equals 5 and K equals 3 to conduct a (5,2)D [ Another aspect of the present invention relates to the above method of conducting a (N,N−K)D GFT NMR experiment where N equals 4 and K equals 2 to conduct a (4,2)D [ Yet another aspect of the present invention relates to the above method of conducting a (N,N−K)D GFT NMR experiment where N equals 5 and K equals 3 to conduct a (5,2)D [ In an alternate embodiment the above method can be modified, where N equals 5 and K equals 3 to conduct a (5,3)D [ Combinations of GFT NMR Experiments A set of multidimensional GFT NMR experiments enables one to devise strategies for GFT NMR-based (high throughput) resonance assignment of proteins or other molecules. Thus, another aspect of the present invention relates to a method for sequentially assigning chemical shift values of an α-proton, Yet another aspect of the present invention relates to a method for sequentially assigning chemical shift values of an α-proton, A further aspect of the present invention relates to a method for sequentially assigning chemical shift values of α- and β-carbons, The present invention also relates to a method for sequentially assigning chemical shift values of α- and β-carbons, Another aspect of the present invention relates to a method for assigning chemical shift values of γ-, δ-, and ε-aliphatic sidechain protons, Yet another aspect of the present invention relates to a method for assigning chemical shift values of γ-, δ-, and ε-aliphatic sidechain protons, A further aspect of the present invention relates to a method for assigning chemical shift values of a γ-carbon, The present invention also relates to a method for assigning chemical shift values of aliphatic and aromatic protons and aliphatic and aromatic carbons of an amino acid residue containing aliphatic and aromatic spin systems in a protein molecule. The method involves providing a protein sample and conducting a set of G matrix Fourier transformation (GFT) nuclear magnetic resonance (NMR) experiments on the protein sample including: (1) a first GFT NMR experiment which is selected from the group consisting of a (5,3)D [ The above-described methods for assigning chemical shift values in a protein molecule can involve further subjecting the protein sample to nuclear Overhauser enhancement spectroscopy (NOESY) (Wüthrich, Another aspect of the present invention relates to a method for obtaining assignments of chemical shift values of The following examples are provided to illustrate embodiments of the present invention but are by no means intended to limit its scope. When designing a GFT NMR experiment ( Acquisition of peaks defining the centers of the chemical shift splittings (“central peaks”) is required for unambiguous assignment if two chemical shift quartets, (Ω GFT NMR data acquisition ( For frequency domain editing, the data sets S The matrices Ĝ(K) and {circumflex over (F)}(K) for time and frequency domain editing of chemical shift multiplets (
The successive identification of peak pairs belonging to central peaks of decreasing order ensures the unambiguous assignment of chemical shift multiplet components ( Depending on the particular magnetization transfer pathway and practical constraints, one can combine the three options for central peak detection. The second and third option offer that (i) magnetization yielding unwanted “axial peaks” in the conventional experiment is used, (Szyperski et al., With respect to Option 1 for central peak acquisition (see Example 2), if the p data sets defining the (N,N
With respect to Option 2 for central peak acquisition (see Example 2), if the basic spectra are recorded with simultaneous acquisition of central peaks from incomplete INEPT, one obtains ε With respect to Option 3 for central peak acquisition (see Example 2), if heteronuclear magnetization is exclusively used for central peak detection, one obtains ε
Table 1 illustrates the representative calculations of the reductions in minimal measurement times in GFT NMR.
For the implementation of (5,2)D For the 76-residue protein ubiquitin nearly all signals of 2D [ With the HACACONHN rf pulse scheme of A 5D FT HACACONHN spectrum acquired with the same maximal evolution times as the basic spectra of (5,2)D HACACONHN GFT NMR SpectrumFirst order central peaks were derived from Ĝ The chemical shift multiplets encoded in the edited spectra B
HACACONHN GFT NMR ExperimentA (5,2)D Because equivalent chemical shift correlations are provided by (5,2)D In order to assess the precision of the chemical shift measurements the resonance line widths need to be considered (Ernst et al., The fact that the individual multiplet components possess the same line widths as the corresponding signals in the parent FT NMR experiment ( Overall, the precision of the indirect shift measurements in the (5,2)D Three different classes of combinations are identified. - (I) 2 combinations provide high precision
- X=
^{1}H^{α},^{13}C^{α},^{13}C′,^{15}N] for all four chemical shifts: - B
**1**[Ω_{0}+Ω+Ω_{2}+Ω_{3}]; B**4**[Ω_{0}Ω_{1}−Ω_{2}+Ω_{3}]; B**6**[Ω_{0}−Ω_{1}+Ω_{2}−Ω_{3}]; and - B
**7**[Ω_{0}+Ω_{1}−Ω_{2}−Ω_{3}], or - B
**2**[Ω_{0}−Ω_{1}+Ω_{2}+Ω_{3}]; B**3**[Ω_{0}+Ω_{1}−Ω_{2}+Ω_{3}]; B**5**[Ω_{0}+Ω_{1}+Ω_{2}−Ω_{3}]; and - B
**8**[Ω_{0}−Ω_{1}−Ω_{2}−Ω_{3}]1 - (II) 26 combinations provide intermediate precision
- X=
^{1}H^{α},^{13}C^{α},^{13}C′,^{15}N] for all four chemical shifts. - (III) 37 combinations provide intermediate precision
- for three of the shifts and low precision [σ=σ(basic)] for one of the four shifts.
The standard deviation depends on the number of equations that need to be linearly combined to calculate the shifts. This can be discussed for three examples, one representing each of the cases. - (I) B
**2**[Ω_{0}−Ω_{1}+Ω_{2}+Ω_{3}]; B**3**[Ω_{0}+Ω_{1}−Ω_{2}+Ω_{3}]; B**5**[Ω_{0}+Ω_{1}+Ω_{2}−Ω_{3}]; and - B
**8**[Ω_{0 -}1- Ω_{2}- Ω_{3}] are selected. Then, the individual chemical shifts are obtained from: - 4Ω
_{0}(“^{5}N)=B**2**+B**3**+B**5**+B**8** -
**4**Ql(^{13}C′)=-B**2**+B**3**+B**5**- B**8** -
**4**Ω_{2}(^{13}C)^{=}B**2**-B**3**+B**5**-B**8** -
**4**Ω_{3}(^{1}H)=B**2**+B**3**- B**5**-B**8** with “BX” representing the shifts extracted from the spectrum BX (X=2,3,5,8). Each shift from BX is associated with a standard deviation of a(basic). Hence, the Gaussian law of error propagation (Eadie et al.,*Statistical Methods in Experimental Physics*, North-Holland, N.Y. (1982), which is hereby incorporated by reference in its entirety) yields:
Thus, the resulting precision is equivalent to the one obtained from four statistically independent measurements. - (II) BI [Ω
_{0}+Qi+Ω_{2}+Ω_{3}]; B**5**[Ω_{0}+Ql+Ω_{2}-Ω_{3}]; B**7**[Ω_{0}+Ω_{1}-Ω_{2}-Ω_{3}]; and B**8**[Ω_{0 - f-Ω}_{2}- Ω_{3}] are selected. Then, the individual chemical shifts are obtained from: - 2 [Ω
_{0}(“^{5}N)=B**1**+B**8** -
**2**Ql(^{13}C′)=B**7**- B**8** -
**2**Ω_{2}(^{13}C)^{=}B**5**- B**7** -
**2**Ω_{3}(H^{1})=BI - B**5** with “BX” representing the shifts extracted from the spectrum BX (X=1,5,7,8). Each shift from BX is associated with a standard deviation of a(basic). Hence, the Gaussian law of error propagation yields:
- (III) B
**1**[Ω_{0}+Ω_{1}+Ω_{2}+Ω_{3}]; B**4**[Ω_{0}−Ω_{1}−Ω_{2}+Ω_{3}]; B**5**[Ω_{0}+Ω_{1}+Ω_{2}−Ω_{3}]; and B**6**[Ω_{0}−Ω_{1}+Ω_{2}−Ω_{3}] are selected. Then, the individual chemical shifts are obtained from: - 2 [Ω
_{0}(^{15}N)=B**4**+B**5** - 2Ω
_{1}(^{13}C′)=B**5**−B**6** - 2Ω
_{2}(^{13}C^{α})=B**1**−B**4**−B**5**+B**6** - 2Ω
_{3}(^{1}H^{α})=B**1**-B**5** with “BX” representing the shifts extracted from the spectrum BX (X=1,4,5,6). Each shift from BX is associated with a standard deviation of σ(basic). Hence, the Gaussian law of error propagation yields:
In case all 15 spectra constituting the constant time (5,2)D GFT NMR experiment are selected, similar considerations show that the resulting standard deviations can be calculated (Eadie et al., (a) Survey of constant time spectra, standard deviations and chemical shift measurements
(b) Calculation of error propagation
The validity of these equations is neatly confirmed by the Monte Carlo simulation performed with input from all 15 spectra:
HACA,CONHN, (5,3)D HACA,CONHN/(5,3)D HACACONHN, and (4,3)D CBCACONHN/(4,3)D CBCA,CONHN GFT NMR ExperimentsThe following GFT NMR experiments were implemented ( (5,2)D (5,3)D (4,3)D HACA,CONHN, (5,3)D HACA,CONHN/(5,3)D HACACONHN, and (4,3)D CBCACONHN/(4,3)D CBCA,CONHN GFT NMR ExperimentsOn a VARIAN Inova 600 MHz spectrometer at 25° C., (i) (5,2)D The yield of peak detection, i.e. the ratio of observed peaks over the total number of expected peaks, was (virtually) complete throughout. Reductions in minimal measurement time, ε, achievable in GFT NMR are given by the ratio of the number of free induction decays (FIDs) of an (N,N−K)D GFT NMR experiment over and the number FIDs of the ND FT NMR experiment. For ubiquitin, the following was obtained: (i) (5,2)D For TT212, the following was obtained: (5,3)D When using (5,2)D Furthermore, the experimental error of chemical shift measurements in constant time GFT NMR experiments scales with 1/(√{square root over (n)}), where n is the number of linear combinations contributing to the determination of a shift (assuming, for simplicity, that the same maximal evolution times have been chosen). The increased accuracy of the measurement is documented by comparing the shifts of the same nuclei measured in intra- and interresidue GFT data. Tables 3 to 5 afford a detailed analysis of the shift measurements associated with sequential connectivities shown in
Automated resonance assignment (Szyperski et al., In principle, with respect to the detection of sequential peaks in the experiments providing the intraresidue connectivities, one may “filter out” the sequential connectivities (e.g., Brutscher, In view of the introduction of cryogenic probes, which reduce NMR measurement times by a factor of 10 or more (Monleon et al., Finally, future research will show to which extent the acquisition speed of GFT NMR can be further increase (Frydman et al., CACBCA, (4,3)D HNN(CO)CACBCA/(4,3)D CBCACA(CO)NHN, (5,3)D HBHACBCACA(CO)NHN, (5,3)D HCC,CH-COSY, (5,3)D HBCBCGCDHD, and (4,2)D HCCH-COSY GFT NMR ExperimentsThe following GFT NMR experiments were conducted for the assignment of polypeptide backbone and sidechain resonances: (i) (4,3)D HNN In the (4,3)D HNN In the (4,3)D HNN(CO) Using the above-described (4,3)D HNN Having obtained the chemical shifts of The information of Resonance assignments of aromatic sidechain spins can be achieved by using a (5,3)D The assignment of the side-chain chemical shifts can be further supported with the (5,2)D HCCCH-COSY experiment. In this experiment for a The radiofrequency pulse schemes for the experiments described in this example are shown in
Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow. Patent Citations
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